Laminin is a large (M.wt=800,000) glycoprotein which is localized to basement membranes, the thin extracellular matrix which separates epithelium from the underlying stroma. Laminin (M.sub.r 900,000) is composed of three chains, A (M.sub.r =400,000), B1 (M.sub.r =210,000) and B2 (M.sub.r =200,000) which are joined by disulfide bonds to form a cruciform-like structure (see FIG. 1). FIG. 1 shows a schematic Model of the laminin molecule. The roman numerals and .alpha. indicate the structural domains. Laminin is of special interest because it has multiple biological activities including promoting cell adhesion, migration, growth, cell differentiation, phagocytosis, collagenase production, tumor cell invasion, and neurite outgrowth. It also binds to many other materials including collagen IV, heparin, heparan sulfate proteoglycan, and entactin. Using proteolytic fragments of laminin, an active site for cell adhesion was proposed to be contained within a large complex formed by the intersection of the three chains. A second active site for cell adhesion and for neurite outgrowth was localized to a region near the end of the long arm. Recently we have identified an active site on the B1 chain near the intersection of the 3 chains. A pentapeptide from the B1 chain sequence (YIGSR) promotes cell adhesion, cell mirgration, receptor binding and reduces tumor metastases in vivo. The abbreviations for amino acids are shown below.
______________________________________ Abbreviations for amino acids Three-letter One-letter Amino acid abbreviaton symbol ______________________________________ Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or aspartic acid Asx B Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glutamine or glutamic acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V ______________________________________
Likewise, the peptide reduces neural crest cell outgrowth from notochord, prevents endothelial cell differentiation, and delays myoblast fusion. It does not promote cell growth, increase collagenase release or promote neurite outgrowth suggesting that these activities lie within another site(s) in laminin.
We have recently cloned and sequenced all of the mouse B1 and B2 chains. Here, we present the isolation and sequence of cDNA clones spanning 9520 bases which encode the entire A chain of mouse laminin. The nucleotide sequence of the clones contains an open reading frame of 3084 amino acids including 24 amino acids of a signal peptide. The A chain contains some eight distinct domains including .alpha.-helices, cysteine-rich repeats and globules.
Accordingly, we have completely cloned and sequenced the A chain of laminin, prepared vectors which express portions of the sequence and identified active peptides. One peptide (PA22-2) is active for cell adhesion, cell growth, haparin binding, neurite outgrowth, collagenase production, cell migration, and increases tumor metastasis and inhibits endothelial cell differentiation. The other peptide (PA21) promotes endothelial cell adhesion and neurite outgrowth. Possible commercial and medical applications include uses as an adhesion and regeneration agent for nerve guides and as an adhesion agent for vascular protheses. The peptide may also be used in wounds and as a control for blood vessel formation. The active peptides have wide usage in research, nerve regeneration and in cancer treatment.
Laminin was first isolated in this laboratory (JBC 254:9933, 1979) and one year later its ability to promote cell attachment was determined here (Cell 22:719, 1980). The entire primary peptide sequence of the three chains was determined from cDNA cloning here. Using synthetic peptides and their corresponding antibodies specific for the B1 chain, we previously identified two active sites of 5 amino acids each which promote cell adhesion, migration, receptor binding and inhibit tumor cell metastases (Graf et al, Cell 48:989, 1987; Graf et al, Biochemistry 26:6896, 1987, Iwamoto et al, Science, 238:1132, 1987, Iwamoto et al, J. Cellul. Physiol. 134:287, 1988 and patent application #7-013,919 which is herein incorporated by reference). We now have recently constructed various vectors in bacterial and mammalian cells which express portions of the A chain protein. We have also identified two active peptides which have some of the biological activities of laminin peptide PA22-2 having 19 amino acids and which promotes cell adhesion, neurite outgrowth, collagenase production, tumor metastases and competitively interferes with endothelia cell differentiation. The second peptide PA21 is 12 amino acids long and it promotes the adhesion of endothelial cells very strongly and has weak activity with other cells. It has neurite outgrowth promoting activity with some neuronal cells when coupled to albumin or transferrin, and can disrupt endothelial cell differentiation.